The coversion of folate to polyglutamate forms by folylpolyglutamate synthetase (FPGS) provides a more effective cofactor for the enzymes of one carbon metabolism and is also required for the cellular retention of folate. The metabolism of folate antogonists to polyglutamate forms is important in the anti-neoplastic efficacy of these agents, and the ability of anti-folates to act as substrates for FPGS is an important factor in the design of new chemotherapeutic agents. FPGS is present in low amounts in eukaryotic cells and may be isolated only with great difficulty. Our goal is to use recombinant DNA methods to force bacteria to synthesize large quantities of the human FPGS enzyme for mechanistic studies and biochemical characterization. We have transfected human DNA into Chinese hamster ovary cells (AUXB1) lacking FPGS activity, and selected for the transfer of the FPGS gene by the ability of cells to grow in media lacking glycine, adenosine and thymidine. Transforming activity appears to be resistant to two six-base restriction enzymes suggesting that the gene is under 10 kb in length. Therefore, we are screening cosmid human genomic libraries for the ability to transfer the FPGS gene to AUXB1 cells. Once identified, the cloned human FPGS gene will be used to obtain a cDNA clone in a high expression plasmid for the production of large quantities of FPGS. The availability of large quantities of the human enzyme will make possible detailed studies on the specificity of the substrate binding sites and on the catalytic mechanism of the enzyme. This will greatly aid in the design of inhibitors of the human enzyme and in the design of antifolates that exert their cytotoxic effects when metabolized to polyglutamate forms.